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1 DOI: 1.138/ncb3342 EV O/E 13 4 B Relative expression shctl Pcdh2_1 C Pcdh2_2 Number of shrns T mterc +/+ G3 mterc -/- D % of GFP + cells Per2 p=.2 p><.1 E % of GFP + cells Pcdh2 p<.1 p<.1 KSL cells w w w G3 mterc -/-, shrn scramble KSL cells w mterc +/+, shrn scramble w w G3 mterc -/-, shrn1 target gene mterc +/+, shrn1 target gene G3 mterc -/-, shrn2 target gene mterc +/+, shrn2 target gene Supplementary Figure 1 Verification of shrns of candidate genes ) The representative Western blot shows the efficiency of knockdown (by shrns) and over-expression (O/E) of Per2. Cell lysates obtained from lineage - bone marrow cells that were infected by lentiviral vectors carrying two different shrn (shrn1 or shrn2) targeting Per2 or Per2-cDN, or an empty control vector were loaded to detect protein expression. Data represent 1 out 3 independent experiments. B) The histogram shows the mrn level of Pcdh2 in lineage - bone marrow cells of WT mice infected by lentiviruses carrying two different shrns against Pcdh2 or a scramble shrn control vector. N= 3 repeat experiments, values are means ± SEM. C) The histogram depicts the number of shrns detected by deep sequencing in the shrn plasmid pool as well as in Lin - cells derived from mterc +/+ and G3 mterc -/- donors after two round of transplantation as depicted in Fig. 1. (D,E) Freshly isolated KSL cells from G3 mterc -/- mice or mterc +/+ mice were infected with 2 independent single shrn constructs targeting (D) Per2 or (E) Pcdh2, or a scrambled shrn control. Infected cells were transplanted into lethally irradiated mice along with non-infected cells from the same culture. The graphs show the changes in the percentage of infected cells (GFP + ) in peripheral blood of primary recipients at indicated time points after transplantation. N= 5 recipients/group, values are means ± SEM, multiple t test was used to calculate p values Macmillan Publishers Limited. ll rights reserved.
2 bsolute number of Ly-HSC per million BM cells p=.36 B bsolute number of My-HSC per million BM cells 2 n.s C CD15 7.5%.5% CD34 6.3% 2.1% Supplementary Figure 2 Per2 deletion improves self-renewal of HSCs in response to replicative stress. -C) Cohorts of 3-month-old mice and mice were sub-lethally irradiated (4 Gy) to induce premature aging and kept for 1 year. t this time point mice were sacrificed to analyze hematopoietic cells in bone marrow (8 mice and 7 mice).,b) The histograms show the absolute number of () Ly-biased HSCs (marked by CD15 lo CD34 - KSL) and (B) My-biased HSCs (marked by CD15 hi CD34 - KSL), values are shown as mean ± SEM, multiple t test was used to calculate p values. (C) Representative FCS plots showing an increase in Ly-biased HSCs (CD15 lo, CD34 - ) in bone marrow of mice vs. mice. FCS was repeated on biological replicates (8 times for mice and 7 times for mice) Macmillan Publishers Limited. ll rights reserved.
3 Percentage of cells scored n.s. _nir _nir _IR _IR Number of gh2x foci per cell B Living cells ( 1 3 ) h p=.1 24h Time after IR p<.1 36h Supplementary Figure 3 Per2 contributes to the activation of apoptosis signaling in hematopoietic cells in response to DN damage but not to DN damage sensing. ) Immunofluorescence staining of gh2x in freshly isolated HSCs (CD34 - LSK) from irradiated (4Gy, 6h after IR) and non-irradiated (nir), 2-3-month-old mice or mice. Representative data derived from 2 analyzed cells for 1 out of 5 mice per genotype are shown. Chi-square test was used to generate p values. Note, there is no impact of Per2 gene status on the induction of gh2x foci. B) 8 freshly isolated KSL cells from 2-month-old and mice were cultured for 12h followed by sublethal irradiation (4Gy). The numbers of living cells were counted 24h and 36h after IR by automatic cell counter. This histogram depicts the number of living hematopoietic stem and progenitor cells (KSL) at indicated time points after sub-lethal irradiation (4Gy). N=5 repeat experiments per genotype, values are shown as mean ± SEM and multiple t test was used to calculate p value Macmillan Publishers Limited. ll rights reserved.
4 B Percentage of cells in PB 8 6 p= IR nir p=.9 IR nir Percentage in BM p=.46 IR nir p=.28 IR nir B22 + CD11b + B22 + CD11b + C nir_per2 41,5% -/- nir_per2 51,2% +/+ 27,3% 2,4% CD11b 42.3% IR_ 64.4% IR_ 25.1% 15.7% B22 Supplementary Figure 4 Per2 deletion rescues myeloid/lymphoid skewing in the context of DN damage. -C) 3-month-old and mice were exposed to 4Gy g-irradiation (IR) or non-irradiated (nir) and were analyzed one year later (same as in Suppl. Fig. 2). (8 mice and 7 mice per group), values are shown as mean ± SEM. Multiple t test was used to calculate p value. () This histogram shows the percentage of B22 + and CD11b + cells in peripheral blood. (B) This histogram shows the percentage of B22 + and CD11b + cells in bone marrow (C) Representative FCS plots showing the B22 + and CD11b + cells in bone marrow of mice of the indicated genotype and treatment Macmillan Publishers Limited. ll rights reserved.
5 Total B22 + CD19 + cells (*1 3 ) ng/ml 2 ng/ml 1 ng/ml Supplementary Figure 5 Per2 deletion does not change B-lymphocyte production of stimulated B cell progenitors from young mice. ) 1 freshly purified EBPs from 2-month-old and mice were cultured and exposed to IL-7 at the indicated concentrations. FCS analysis determined the total numbers of B22 + CD19 + cells after 4-day culture. N= 5 mice per group, dots represent individual mice, lines depict mean values. In contrast to the results on EBPs from old mice (Figure 5B), Per2 gene status does not affect B-lymphocyte production rates of stimulated B-cell progenitors from young mice Macmillan Publishers Limited. ll rights reserved.
6 13KD 13KD Suppl. Fig. 1 Fig. 3B 5KD p-chk1 13KD Fig. 2F Fig. 3C p-tm (S1981) 2KD 13KD 55KD 55KD CHK2 Fig. 3G 55KD Fig. 2G p-p53 (S15) 13KD PUM p21 BCL2 BX PUM 15KD 55KD cleaved CSP3 BX p-p53 p21 15KD cleaved CSP3 BCL2 Fig.2H Fig. 3K Supplementary Figure 6 Unprocessed photographs of bigger sections of the Western blots with size markers corresponding to the indicated Western blots in the main figures. Black squares indicate cutting of Western blots as depicted in main figures. Western blots for detected proteins were run in parallel with control blots with the same loading and running order Macmillan Publishers Limited. ll rights reserved.
7 7KD E47 Fig. 6 13KD 15KD BTF Fig. 7B Fig. 7C Supplementary Figure 6 continued Macmillan Publishers Limited. ll rights reserved.
8 Supplementary Table Legends Supplementary Table 1 Details of shrn screening Supplementary Table 2 RT-PCR primers Macmillan Publishers Limited. ll rights reserved.
MLN8237 induces proliferation arrest, expression of differentiation markers and
Supplementary Figure Legends Supplementary Figure 1 827 induces proliferation arrest, expression of differentiation markers and polyploidization of a human erythroleukemia cell line with the activating
Supplementary Figures
Supplementary Figures Supplementary Figure 1: Phenotypically defined hematopoietic stem and progenitor cell populations show distinct mitochondrial activity and mass. (A) Isolation by FACS of commonly
(A-B) P2ry14 expression was assessed by (A) genotyping (upper arrow: WT; lower
Supplementary Figures S1. (A-B) P2ry14 expression was assessed by (A) genotyping (upper arrow: ; lower arrow: KO) and (B) q-pcr analysis with Lin- cells, The white vertical line in panel A indicates that
Supporting Information
(xe number cells) LSK (% gated) Supporting Information Youm et al../pnas. SI Materials and Methods Quantification of sjtrecs. CD + T subsets were isolated from splenocytes using mouse CD + T cells positive
88.7 <GFP-A>: EGFP GFP. Null. % of Cells! <APC-A> B220!
B. C. 1 Cell Number (X18) A. % of Cells % of Max 8 Con 6 4 88.7 1 13 14 : EGFP 4 6 4 1 WT 8 % of% ofcells Max 6 6 4 G. 1 13 B Hdac3-/- 14 15 1 13 14 15 Ter119 Ly6C Wild type WT 4
Flow cytometry Stained cells were analyzed and sorted by SORP FACS Aria (BD Biosciences).
Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice were used for LSK transduction and competitive repopulation assays. Animal care was in accordance with the guidelines of Keio University for animal and recombinant
Supplemental Information Inventory
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show PB hemoglobin concentration (Hgb) and platelet counts (Plt) in Kras ex3op/ex3op mice
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Supplementary Fig. 1: Characterization of Asxl2 -/- mouse model. (a) HSCs and their
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(a) Scheme depicting the strategy used to generate the ko and conditional alleles. (b) RT-PCR for
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Supplemental Table S1. RT-PCR primers used in this study
Supplemental Table S1. RT-PCR primers used in this study -----------------------------------------------------------------------------------------------------------------------------------------------
administration of tamoxifen. Bars show mean ± s.e.m (n=10-11). P-value was determined by
Supplementary Figure 1. Chimerism of CD45.2 + GFP + cells at 1 month post transplantation No significant changes were detected in chimerism of CD45.2 + GFP + cells between recipient mice repopulated with
Supplementary Methods
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Supplementary Figure 1 Schematic and results of screening the combinatorial antibody library for Sox2 replacement activity. A single batch of MEFs were plated and transduced with doxycycline inducible
We designed the targeting vector in such a way that the first exon was flanked by two LoxP sites and
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SUPPLEMENTRY INFORMTION DOI:.38/ncb Kdmb locus kb Long isoform Short isoform Long isoform Jmj XX PHD F-box LRR Short isoform XX PHD F-box LRR Target Vector 3xFlag Left H Neo Right H TK LoxP LoxP Kdmb Locus
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Supplementary Figure 1. Nature Structural & Molecular Biology: doi: /nsmb.3494
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Best practices in panel design to optimize the isolation of cells of interest
Sort Best practices in panel design to optimize the isolation of cells of interest For Research Use Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor is a registered trademark of Life
* * IgL locus (V - J ) λ λ
Supplementary Figures and Figure Legend Supplementary Figure S1 A C F Vav-P-BCL2 doner mice 8 6 4 2 Lymphoma free (%)1 Tumor Pathology Score Retroviral transduction Vav-P-BCL2 HPCs HPC + shrna VavP-Bcl2
supplementary information
DOI: 10.1038/ncb1977 Figure S1 a. Immunofluorescence analysis of IFT20 localization in PBL costained with anti-β-tubulin antibodies. b. Immunofluorescence analysis of IFT20 localization in Jurkat cells,
Supplementary Figure 1. Homozygous rag2 E450fs mutants are healthy and viable similar to wild-type and heterozygous siblings.
Supplementary Figure 1 Homozygous rag2 E450fs mutants are healthy and viable similar to wild-type and heterozygous siblings. (left) Representative bright-field images of wild type (wt), heterozygous (het)
Measurement of peritoneal macrophage apoptosis by Celigo plate imaging cytometer
SUPPLEMENTAL METHODS Measurement of peritoneal macrophage apoptosis by Celigo plate imaging cytometer For Celigo experiments, 0.1 ml containing 5 x 10 4 cells was seeded into 96 well plates for 30 min
DC enriched CD103. CD11b. CD11c. Spleen. DC enriched. CD11c. DC enriched. CD11c MLN
CD CD11 + CD + CD11 d g CD CD11 + CD + CD11 CD + CD11 + Events (% of max) Events (% of max) CD + CD11 Events (% of max) Spleen CLN MLN Irf fl/fl e Irf fl/fl h Irf fl/fl DC enriched CD CD11 Spleen DC enriched
Revised: RG-RV2 by Fukuhara et al.
Supplemental Figure 1 The generation of Spns2 conditional knockout mice. (A) Schematic representation of the wild type Spns2 locus (Spns2 + ), the targeted allele, the floxed allele (Spns2 f ) and the
SUPPLEMENTARY INFORMATION
a before amputation regeneration regenerated limb DERMIS SKELETON MUSCLE SCHWANN CELLS EPIDERMIS DERMIS SKELETON MUSCLE SCHWANN CELLS EPIDERMIS developmental origin: lateral plate mesoderm presomitic mesoderm
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DOI: 10.1038/ncb2271 Supplementary Figure a! WM266.4 mock WM266.4 #7 sirna WM266.4 #10 sirna SKMEL28 mock SKMEL28 #7 sirna SKMEL28 #10 sirna WM1361 mock WM1361 #7 sirna WM1361 #10 sirna 9 WM266. WM136
Antibodies used in this study Figure S1. Akt expression in neutrophils from WT and individual Akt isoform knockout mice
ntibodies used in this study The anti β-actin monoclonal antibody (Sigma-ldrich, St. Louis, MO) was generated against a slightly modified human β-actin N-terminal peptide, c-sp-sp-sp-ile-la-la-leu-val-ile-
Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 PPAR-γ is dispensable for the development of tissue macrophages in the heart, kidneys, lamina propria and white adipose tissue. Plots show the expression of F4/80 and CD11b (a) or
TIB6 B16F1 LLC EL4. n=2 n=7 n=9 n=6
Shojaei et al., Supplemental Fig. 1 9 Mean Inhibition by anti-vegf (%) 7 5 3 1 TIB6 n=2 n=7 n=9 n=6 Supplemental Figure 1 Differential growth inhibition by anti-vegf treatment. The percent of tumor growth